Particle based assay system

ABSTRACT

The invention provides a particle comprising a surface, wherein a portion of the surface is capable of emitting electromagnetic radiation and another portion of the surface is capable of emitting a differential electromagnetic radiation (either different intensity, different frequency or no radiation), and wherein the arrangement of said portions of the surface defines a spatially distributed code for identifying the particle. The invention also provides method of manufacturing a particle having an identifying code comprising providing a particle with a functionalized surface which comprises functional binding moieties and selectively removing a plurality of the functional binding moieties from the surface to create a pattern of functionalized and differentially-functionalized zones on the surface. Various liquid-based assay methods employing the particle of the invention and a kit comprising the particle of the invention are also disclosed.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/284,706, filed Apr. 18, 2001. The contents of that application in itsentirety are hereby incorporated by reference into this specification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to devices and methods forscreening, selecting, and validating small molecule and biologic drugcandidates in solution and, more particularly, to spatially distributedidentification tags that facilitate the direct quantitative,semi-quantitative, or qualitative assay of proteins, genes and theirbiologic products (carbohydrates, lipids or other cellular components).

2. Related Art

All patents and publications cited throughout the specification arehereby incorporated by reference into this specification in theirentirety in order to more fully describe the state of the art to whichthis invention pertains.

The mapping of the human genome, currently believed to comprise of somethirty thousand genes, has led to an exponential growth in dataavailable to pharmaceutical companies. The linkage between specificgenes and disease processes, namely functional genomics, will, it isbelieved, provide a means of better screening small molecule librariesagainst druggable genes (genes that are believed to be functionallyrelated to a specific disease). Small molecules will be selected on thebasis of their ability to influence the expression profile of themessenger RNA (mRNA) of such genes. There are, however, limitations tosuch selection strategies. Firstly, the strategy assumes that all geneproducts are known and can be assayed; secondly, the strategy assumes aone to one relationship between gene and protein. The occurrence ofgene-splice variants in all but infectious diseases and prokaryoticspecies belies the former assumption, whilst post-translationalmodification (e.g., glycosylation, phosphorylation, acetylation) ofproteins as well as environmental parameters obviate the latterassumption. As a consequence of these limitations, proteomics, whose aimis the mapping of proteins, protein-protein interactions, and theirmetabolic, catabolic, and anabolic pathways, has grown in significanceas a means of facilitating the selection of small molecules, andbiologics (e.g., protein therapeutics, monoclonal antibodies, vaccines,therapeutic serum or gene transfer products), against a growing numberof established, and yet to be established, protein targets. The emergingfield of proteomics is estimated to yield in excess of 10,000 proteinsduring this decade. Identification and validation of these potentialtargets will require substantial equipment, supplies for testingin-vitro with cell based assays, in-vivo with animal models andeventually with human clinical trials in order to ensure drug discoveryand subsequent development.

Such testing requires small molecules or biologics to be screenedagainst samples of tissue and physiological fluids, comprising theprotein, genes, or other biological (carbohydrate, lipid) targets ofinterest. Such samples can be costly and difficult to access, oftenrequiring a priori confirmation that the testing to be conducted istherapeutically relevant and justified. A blood sample likely containsgreater than 10,000 protein targets, yet existing instrumentation andassay methodology limits the number that can be realistically used astargets to a far smaller figure. A pharmaceutical company will typicallyscreen all of its small molecule against less than one percent of thisfigure, necessarily eliminating many potential drug targets, andpossible drug candidates.

This strategy requires sophisticated instrumentation that can purifydrug leads, screen the vast number of leads with their protein targets,and analyze and interpret the results. The primary methods usedtypically involve each or a combination of the following: liquidchromatography (LC) an expensive but well established method primarilyfor the distillation of drug leads, 2d (Isoelectric Focusing) and 1d-gelelectrophoresis (SDS-PAGE), yeast 2-hybrid systems, mass spectrometry(MS), and various types of immunoassay. All of these methods involvewell established but costly instrumentation, requiring specialistexpertise for their operation and interpretation of data. These methodsdo not provide, either individually or collectively, a method of rapidlyscreening a massive number of small molecules or biologics against anequally massive number of protein targets. This is, in part, due to thefact that not all protein targets are known. 2d-gel electrophoresis isthe primary method for the mapping out of proteins (estimated at between60,000 to 150,000 per mammal) but has resolution limits imposed by thedistance between spots and the protein loading per spot, thussignificantly limiting the technique to high abundance proteins. This isa severe limitation given that many low abundance proteins are believedto play key roles in cellular signaling and disease pathways, and thatprotein activity provides more therapeutically valuable information thanprotein abundance. Moreover proteins often do not act alone. Anincreasing effort is being spent on examining how proteins interact, notonly with other proteins but also with nucleic acid, small molecules andligands. A current popular method is to use antibodies as capturemolecules to trap interacting proteins. The immunoprecipitate is thenrun out on a 1-D gel, digested and analyzed by tandem MS to determinethe identity of the interacting partners. Yeast 2-hybrid systems arepowerful tools for the identification of protein-protein and protein-DNAinteractions, although they are hampered by high rates of falsepositives, a poor ability to identify weak interactions, a relativelylow throughput and are not suited generally to the study ofprotein-ligand interactions. MS requires “clean” samples and is not goodat analyzing protein complexes. Immunoassays provide a means ofdetermining the kinetics and cross-reactivities associated with thebinding of drug targets to drug compounds. They are usually conducted inmicrotiter plates of either 96-well or 384-well format. However, theserial nature of this process combined with the requirement for washing,incubation, and heavy reagent consumption, mean that this is a costlyand time-consuming process.

There is a strong drive for technologies that facilitate (i) the costeffective identification of proteins and their interactions with otherproteins, as well as the role they play in metabolic, catabolic, andanabolic pathways; (ii) the cost effective profiling of proteins interms of abundance and/or activity; (iii) the cost effective screeningof massive numbers of small molecules and biologics against selectedproteins.

Such technologies typically require a combination of speed, low reagentand sample consumption, multiplexing (i.e., analysis of multiple targetsin parallel), low cost (particularly relating to any disposableelements), high assay repeatability, robust biochemical surfaces, andhigh sensitivity & selectivity. These requirements resulted in thedevelopment of protein microarrays which provide a means of massproducing surfaces, of typically a few centimeters square, comprising ofa massive number of multiple target probes, usually proteins that arespecific to, and bind to, known target proteins such as monoclonalantibodies (MABs). Much of this technology has evolved from genemicroarrays. However, in contrast to gene arrays, where probes aretypically synthetic oligonucleotides, protein microarrays suffer anumber of important disadvantages: denaturation of complex proteinstructure due to either the protein attachment process and/or storageconditions; sensitivity and selectivity, due to the affinity andcross-reactivity of the binder protein used; and cost, due to the natureof the mass manufacturing technology used, often based on either siliconor a special glass. Furthermore, relative to the in situ synthesis ofoligonucleotides, specific to target DNA/RNA, and used for thesequencing of DNA, identification of mutations (such as singlenucleotide polymorphisms, SNPs), proteins cannot be built up in such away. In situ synthesis of amino acids has been attempted but without anycommercial success to date and needs to be added to a surface in apreformed fashion, i.e. as complete antibodies, mimics, or other form ofbinder protein. This necessarily limits the speed at which the processcan be achieved, increases the costs, and requires access to such binderproteins.

In all cases, conventional approaches rely on a priori knowledge oftarget proteins and pathways in order to develop binder proteins, thatform the basis of the biochemical probe arrays used to query thosetargets, and gain information on, for example, drug (small molecule,biologic) efficacy and toxicity.

The in situ synthesis of oligonucleotide arrays onto silicon substratesvia the use of photo-labile groups and a series of masking and demaskingsteps, allowed Affymetrix to develop and produce its GeneChip™. Thistechnology has provided a method of mass-producing such arrays onsilicon using technology largely inherited from the semiconductorindustry. These chips are, however, expensive, require lead times of upto one month, and provide oligomers of a limited number of bases. Theyalso require a priori knowledge of the target genes. The length of theoligomers is limited by the photoactivation process used. It means thatyield would be very poor for oligomers of greater than 25 bases inlength. This significantly limits the sensitivity of this method forapplication such as gene-expression profiling, where only abundant genesare detected and not low copy numbers of genes, or in some cases theirsplice variants. This shortcoming can be circumvented by PCRamplification of the expressed RNA, however, artifacts are known to becreated by such processes along with the fact that the biases introducedby PCR must be accounted for in the interpretation of results and geneexpression profiling methodology used. The limited flexibility and highaccess costs associated with Affymetrix's technology have resulted in anumber of companies, and users, producing glass-slide based arrays. Thishas been due in part to the increasing availability of so-calledarrayers and spotters that, by various dispensing methods (e.g.,ink-jets and pins) can deposit oligonucleotide or proteinprobe-containing reagents onto various substrates, and using a varietyof surface chemistries and functional groups (e.g., amines andaldehydes, and the like) attach these probes to the surface. One methoddeveloped by Rosetta provides an efficient method of ink-jet printingnucloetide bases onto a substrate, which are subsequently in situsynthesized using conventional phosphoramidite chemistry, which does notsuffer the aforementioned shortcomings of the Affymetrix approach.

Despite significant growth in the use of these microarray-based systems,limits in the applicability of these systems have led to dissatisfactionamong users and many pharmaceutical companies have expressed interest inalternative methods of achieving high throughput screening.

Bead-based assays have therefore been developed which overcome thelimitations of the microarray technology. The superior mixing in abead-based array results in negligible mass transfer of target to bead,as opposed to microarrays where target diffusion is always mass-transferlimited. This results in faster time-to-result, reduced need forwashing, and improved signal to noise ratios. Bead-based arrays alsoallow for greater spatial independence relative to microarrays, whereprobes occupy a fixed position on a substrate and cannot be individuallymanipulated. Such advantages are not only of interest to the trackingand manipulation of compounds in combinatorial libraries, but also toassays for application in diagnostics, prognostics, and drug discovery.

Luminex has developed a particle-based assay format employingmicron-scale microspheres, whose coding is achieved through the mixingof two different fluorochromes (incorporated into polystyrene particles)in different molecular weight ratios. See, e.g., U.S. Pat. Nos.6,268,222 and 5,736,330. Luminex has achieved 64 different codes by thismethod. A higher number of codes would require the use of 3 or moredifferent fluorochromes. Spectral discrimination of codes becomes morechallenging as do the costs associated with manufacturing the particles.Some coding schemes employ fluorescent spectra as a means ofdistinguishing particles. This can present a problem in media wherebackground fluorescence occurs in the same frequency range as thecoding. Such a situation would include the assay of various proteins inwhole blood. Alternative approaches, currently under development,include that of Quantum Dot whose coded particles are distinguished byvery narrow symmetric emission spectra, obtained by the nanometrictuning of semiconductor nanocrystals. See, e.g., U.S. Pat. No.6,274,323. Also, SurroMed, discloses particles that are electroplatedinto the pores of an alumina membrane to which a silver electrode hasbeen evaporated. See, e.g., WO 01/02374 and WO 00/65472. In the case ofSurroMed, metals exhibiting different reflectivities areelectro-deposited into these pores.

The codes are provided by differential reflectivity. However, thesetechnologies have limitations in practice, including the fact thatattachment of proteins on semiconductor nanocrystals is non-trivial andtends to denaturation, and the utilization of metal substrates (such asby SurroMed) facilitates non-specific adsorption of non-target proteins,as well as limitations in their applicability such as the fact thatpresenting a reflectance-based code on a particle would be difficult toread in turbid media such as whole blood.

Finally, particle-based assay formats are typically run through flowcytometer instruments. Most of the above described particle basedformats, however, require customized cytometers due to the need todetect optical emissions at different wavelengths to those that resultsolely from the binding of, for example, an antibody to an antigen (e.g.an antibiotic), and the subsequent attachment of a reporter antibody, towhich is attached a fluorophore. Conventional sandwich immunoassayinvolves the washing of beads to which antigen and reporters have boundfollowed by excitation of the bead by a suitable wavelength source suchthat binding events could be detected, and in the case of adose-response assay, quantitation of analyte measured at a specificpoint in time following exposure of antibody to antigen, through therelationship between emission intensity and analyte (target)concentration.

SUMMARY OF THE INVENTION

This invention provides a particle comprising a surface, wherein aportion of the surface is capable of emitting a first electromagneticradiation and another portion of the surface is capable of emitting adifferential electromagnetic radiation, and wherein the arrangement ofsaid portions of the surface defines a spatially distributed code foridentifying the particle. In the practice of the invention, thedifferential electromagnetic radiation may comprise electromagneticradiation of a different intensity or a different frequency than thefirst electromagnetic radiation or can be no electromagnetic radiationat all.

This invention further provides a method of manufacturing a particlehaving an identifying code comprising providing a particle having afunctionalized surface which comprises functional binding moieties andselectively removing a plurality of the functional binding moieties fromthe surface to create a pattern of functionalized anddifferentially-functionalized zones on the surface.

This invention also provides a method of manufacturing a particle havingan identifying code comprising providing a particle having adifferentially-functionalized surface and selectively adding a pluralityof functional binding moieties to the surface to create a pattern offunctionalized and differentially-functionalized zones on the surface.

The invention further provides a particle comprising a surface whereinthe surface comprises at least one modified portion comprising aplurality of functional binding moieties and at least one unmodifiedportion which is substantially free of functional binding moieties,wherein the modified portion(s) further comprise an electromagneticradiation emitting species, and wherein the arrangement of modified andunmodified portions on the surface forms a pattern amenable todetection.

The invention also provides an article of manufacture having asubstantially cylindrical shape with a diameter between about 5 μm and200 μm, and a length between about 10 μm and 2000 μm, wherein thesurface of the particle comprises at least one substantiallycircumferential functionalized zone, at least one substantiallycircumferential differentially-functionalized zone between the end ofthe particle and the functionalized portion or between functionalizedportions, and a combination of the width(s) of the functionalizedportion(s) and the width(s) of the differentially-functionalized zone(s)establishes a code for identifying the particle.

The invention further provides a labeled oligonucleotide librarycomprising a plurality of oligonucleotide compounds attached to aplurality of particles of the invention. In certain embodiments, eachparticle of the labeled oligonucleotide library contains a singlespecies of oligonucleotide compound. The oligonucleotide compounds canbe naturally-occurring, synthetic or semi-synthetic oligonucleotides.

The invention also provides a method of determining the sequence of anunknown oligonucleotide species in a solution comprising providing alabeled oligonucleotide library of the invention, wherein theoligonucleotide sequence of each oligonucleotide attached to eachparticle in the library and the characteristic electromagnetic emissionintensity profile of each particle in the library is stored andcorrelated in a database, mixing the solution with the labeledoligonucleotide library under conditions sufficient to permit theunknown oligonucleotide speices to hybridize with correspondingparticles of the lableled oligonucleotide library, exciting theparticles to produce the characteristic electromagnetic emissionintensity profile corresponding to the particle(s) in the library,detecting the characteristic electromagnetic emission intensity profileand correlating it to the characteristic emission intensity profile inthe database, thereby determining the sequence of the unknownoligonucleotide species.

The invention further provides a method for determining the amount of atarget compound in a test sample comprising the steps of incubating amixture of a test sample suspected of containing the target compoundwith at least one particle according to the invention wherein theparticle(s) comprise a plurality of an antibody which is specific forthe target compound, under conditions appropriate to formtarget/antibody complexes, wherein the electromagnetic radiationemission occurs upon formation of the target/antibody complex, andmeasuring the amount of electromagnetic radiation present in saidmixture thereby determining the amount of target compound in said testsample.

The invention also provides an assay for the detection of bindingbetween a probe and a target comprising contacting the probe with asolution suspected of containing the target, wherein the probe isattached to a particle which emits electromagnetic radiation uponbinding between probe and target and emits electromagnetic radiation asa means of identifying the particle in solution.

Finally, the invention provides a kit for the detection of bindingbetween a probe and a target comprising at least one particle of theinvention and a flow cytometric device.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(A) to 1(D) illustrates an example of the process of extrusion,functionalization and coding of the surface of a filament, and itssubsequent singulation into individual particles

FIG. 2 illustrates a preferred embodiment of a particle based assaymethod of the invention for a biomarker assay.

FIG. 3 illustrates a preferred embodiment of a particle based assaymethod of the invention for a DNA assay.

FIGS. 4(A) and (B) illustrate the surface functionalization of theparticle material according to the invention. FIG. 3(A) shows apolypropylene material surface prior to plasma treatment and FIG. 3(B)shows the polypropylene material surface following plasma treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to particle tags useful foridentifying molecules in liquid-based assay systems. Each particle has asurface comprising a plurality of functional binding moieties inselected areas, such that the surface comprises zones of functionalizedmaterial interspersed with zones of differentially-functionalizedmaterial. The functionalized zones further comprise a moiety that emitselectromagnetic radiation, typically upon reaction or excitation fromanother source of energy. The functionalized anddifferentially-functionalized zones are spaced in such a way on theparticle surface so as to create a pattern providing a spatiallydistributed code for identifying the particle. The code is “read” bydetecting the pattern of electromagnetic radiation emitted in thefunctionalized zones. The code can be stored in a database forcomparison with data from tracked particles in an assay to determine theidentity of a molecule attached to the functional binding moiety on aparticle. The electromagnetic radiation emitting moiety may be attacheddirectly to the particle in the functionalized zones or may be attachedto the molecule attached to the functional binding moieties in thefunctionalized zones.

In a preferred embodiment, the particle is formed in the shape ofcylinder of polymeric material and the coding of the particle is in theform of a series of bands of functionalized binding moieties alternatingwith bands containing non-functionalized binding moieties. In apreferred manufacturing method, the polymeric material is extruded as afilament (FIG. 1A), functionalized by a plasma treatment (FIG. 1B),selectively ablated to create the non-functionalized bands (FIG. 1C),and singulated or cut into pieces, (FIG. 1D) to create the cylindricalparticles with a spatially distribute code for identifying eachparticle.

As explained in detail herein, a catalog or library of particles can beused to track and/or identify target compounds in solution byidentifying the particle to which a probe and electromagnetic radiationemitting species is attached when a binding event between the probe andthe target causes the electromagnetic radiation emission. The particlesof the invention can be used to identify unknown samples comprising, forexample, a fluorophor-labeled protein or nucleic acid. In the case of anassay for detecting a protein target suspected of being in solution, theprobe may comprise a known receptor for the protein and afluorophor-labeled secondary antibody could also be added to the samplefor the purposes of a sandwich assay. In the case of an unknownnucleotide sequence, a fully degenerate set of fluorophor labeledcomplementary nucleotide probes may be used to detect a binding eventthat would trigger emission of the fluororophor. After being mixed withthe unknown protein or nucleic acid in the sample, the particles areflowed past a suitable light source (e.g. a laser). If any of theprotein or nucleic sample binds with a corresponding antibody/mimic oroligonucleotide sequence probe attached to one of the coded particles,the fluorophore will give off energy at each of the functionalized zonesof the particle, which can then be detected by, for example, a suitableoptical reader system (e.g. PMT, CCD). Because each peak of energy fromeach functionalized zone will be separated by a particular distance froma differentially-functionalized zone, the detector produces a resultantwaveform for each particle. Each particle will have a spatiallydistributed pattern of functionalized and differentially-functionalizedzones, the pattern being determined by, for example, the width andspacing of such zones. A computer can be employed to compare thedistances between the peaks of the waveform detected in the sample, tothe waveform of cataloged particles to identify the particular catalogedparticle which has bound to the sample. Once the particle is identifiedin the database, the identity of the unknown protein or nucleic acidwithin the sample is determined.

I. Particle Tags.

This invention provides a particle comprising a surface, wherein aportion of the surface is capable of emitting a first electromagneticradiation and another portion of the surface is capable of emitting adifferential electromagnetic radiation, and wherein the arrangement ofsaid portions of the surface defines a spatially distributed code foridentifying the particle. As used herein, the term “differentialelectromagnetic radiation” can mean, for example, electromagneticradiation of a different intensity or different frequency than the firstelectromagnetic radiation or can be absence or near absence ofelectromagnetic radiation. In a preferred embodiment, the differentialelectromagnetic radiation is the absence or near absence ofelectromagnetic radiation. The particles of the invention are typicallyformed from a mass of material. The material preferably has a specificgravity such that the particles formed there from are iso-buoyant in thecarrier fluid used in, for example, a flow cytometer (comparable to thedensity of water; i.e., 1.0 g/cm³). Such a density avoids particlescreated from the material from sedimenting out or floating. This densityalso allows the particles to maintain lateral flow in a flow cytometer'sliquid-handling system or any similar system used to track and identifythe particles. Moreover, the material should include suchcharacteristics that result in particles that are non-aggregating.Specifically, the resulting particles should not aggregate or formclusters since clustered particles would create difficulty indistinguishing one particle from another. Further, the particles shouldbe resistant to attractive forces such as electrostatic charge andideally have a surface which is highly inert in its natural state,unless activated. In addition, the particle material preferably has alow intrinsic electromagnetic radiation emission, so as to reduceinterference with the electromagnetic radiation of the coding andmaximize the signal to noise ratio.

Preferred examples of the types of materials having the above-identifiedproperties that can be formed into particles according to the presentinvention include materials chosen from the group consisting ofpolymers, composites, inorganics, natural products, and combinationsthereof. The polymer material useful in the present invention cancomprise electrically non-conducting or conducting polymers. Examples ofacceptable polymeric materials include but are not limited topolystyrene, halogenated polystyrene, polyaniline, polyacetylene,polypyrrole, polyacrylic acid, polyacrylonitrile, polyamide,polyacrylamide, polyacrolein, polybutadiene, polycaprolactone,polycarbonate, polyester, polyethylene, polypropylene, polyethyleneterephthalate, polydimethylsiloxane, polyisoprene, polyurethane,polyvinylacetate, polyvinylchloride, polyvinylpyridine,polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride,polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide,poly(lactide-co-glycolide), polyanhydride, polyorthoester,polyphosphazene, polyphosophaze, polysulfone, as well as cross-linkedpolystyrene such as with divinylbenzene, grafted copolymers such aspolyethyleneglycol/polystyrene, dimethylacrylamide, which can also becross-linked such as with N,N′-bis-acryloyl ethylene diamine, and anycombinations thereof. Examples of “natural products”, as used herein,include such material as carbohydrate, including carboxymethylcellulose, hydroxyethyl cellulose, agar, gel, proteinaceous polymer,polypeptide, lipid, metal, resin, latex, rubber, silicone, e.g.,polydimethyldiphenyl siloxane, glass, ceramic, charcoal, kaolinite,bentonite, silk, wax, rubber, resins, and the like. Examples of“composites” are those such as glass fiber composites, carbon fibercomposites, and combinations thereof. Examples of “inorganic” materials,as used herein, include inorganic polymers, metal oxides (e.g., silica,alumina), mixed metal oxides, metal halides (e.g., magnesium chloride),minerals, quartz, zeolites, TEFLON, and the like. General reviews ofuseful materials for forming particles that include a covalently-linkedreactive functionality can be found in Atherton et al., Prospectives inPeptide Chemistry, Karger, 101-117 (1981); Amamath et al., Chem. Rev.,77:183-217 (1977); and Fridkin, The Peptides, Vol. 2, Chapter 3,Academic Press, Inc., (1979), pp. 333-363.

Most preferred of these materials are polymeric materials. To create theparticles from polymer material, the material is preferably firstextruded, and then cold drawn, to produce a fine filament having agenerally cylindrical cross-section with a predetermined diameter.Diameters of polymeric particles formed this way range generally between1 μm and 1000 μm, and preferably between 10 μm and 50 μm, and mostpreferably approximately 20 μm.

Alternative methods of manufacture such as LIGA, hot (or UV) embossing,casting or injection moulding could also be employed to manufacture theparticles of the invention. LIGA (Lithographie, Galvanoformung andAbformung, i.e. lithography, electroplating and moulding) is a threestage process which can be used for the manufacture of high aspectratio, 3-D microstructures in a wide variety of materials, includingpolymers. Irradiating a resist (polymer) is the first step in theprocess. This can be achieved using laser light, electron or ion beamsor X-rays from a synchrotron radiation source, the latter beingessential for deep structures. Preferably, these methods use a shadowprinting process. In the deep X-ray lithographic process a 2-D absorberpattern from a mask is transferred into the depth of a thick resist bythe chemical changes induced by a highly collimated beam of X-rays.Development of the irradiated areas of the resist results in a 3-Dreplication of the pattern and a metallic master mould can then beproduced by electroplating into the ‘free’ areas. In this variant of themethod of making the particles of the invention, separate layers ofpolymer are deposited onto a flat substrate “master” such that cylindersare built up comprising “plasma functionalizable” and “nonplasma-functionalizable” regions. The plasma non-functionalizableregions comprise a normally inert surface, such that on exposure to aplasma treatment process, only the plasma-functionalizable regions arefunctionalized. This could be achieved through the use of a photoresistwith an optically modifiable surface or one whose starting surface is100% occupied with a different functional group, thus is not conduciveto further functionalization. These groups are effectively saturated (ifnecessary) by a process that does not effect thefunctionalizable/functionalized surface. In this instance both polymericmaterials would exhibit a low auto-fluorescence.

Hot or UV embossing involves the transfer of structures, typically froman electroformed Nickel shim, into appropriate polymer materials (suchas polycarbonate, PMMA, polystyrene). A further variant on thefabrication of the particles of the invention would modify polymericshapes (e.g., cylindrical or rectangular) on a flat substrate by eitherheat or UV so as to remove or render inactive pre-defined areas of thosesurfaces (defined by the Nickel shim), following a plasma treatmentactivation stage.

Injection moulding, LIGA, or UV/hot embossing, or casting, could be usedto mass produce many cylinders, or other appropriate polymer shapes, inparallel, that could then be plasma treated and selelctivelyfunctionalized with a UV-excimer laser.

The particle of the invention typically has a shape formed by designedextrusion die or can be free form. Preferred shapes include, but are notlimited to, cylindrical, spherical, conical, elliptical, bar-like,slab-like, ribbon-like, ovoid, spiral, amoeba-like, or tube-like. Theshapes are preferably solid but can be to a certain extent hollowprovided they retain the general characteristics noted above. Preferredof these shapes are those having width and length dimensions that allowfor the particle to have a surface that can be manipulated as describedherein to provide the spatially distributed code. In one embodiment, theparticle of the invention has an aspect ratio, of width to length, offrom about 1:2 to about 1:10. In a preferred embodiment the particle hasan aspect ratio of width to length of from about 1:3 to about 1:5. In analternative embodiment, the particle is generally a sphere, i.e., havingan aspect ratio of approximately 1:1. The aspect ratio chosen willsubstantially determine the proper horizontal orientation of theparticle in its lateral flow through a detection instrument, for examplethe fluidic channel of a cytometer's detection head. The surface of theparticle can be flat, curved, rough, smooth, or any combination thereof.

II. Electromagnetic Radiation.

Each of the particles formed according to the invention contains aspatially distributed code created by the arrangement on the surface ofthe particle of portions or areas or zones that emit a firstelectromagnetic radiation among portions or areas or zones that emit adifferential electromagnetic radiation, i.e., electromagnetic radiationof, for example, a different frequency, a different intensity, orsubstantially no radiation. As explained in more detail below, thedifferent zones are created on the surface of the particle bymodification or functionalization of the surface to allow for selectiveattachment of sample molecules bearing electromagneticradiation-emitting moieties or for attaching the electromagneticradiation-emitting moieties directly to the surface. In a preferredembodiment, the electromagnetic radiation emitted by such moieties has awavelength from infrared to ultraviolet so as to allow for detection byrelatively inexpensive and widely available electromagnetic sensingmeans. Within this range, the preferred wavelengths are between 400 nmand 1 μm.

The electromagnetic radiation emitted by the moieties attached to theparticles can be derived from optical or non-optical excitation of themoiety or combinations thereof. Examples of non-optical excitationinclude, but are not limited to, electrical, chemical, biological,electrochemical and combinations thereof. Preferred moieties that emitelectromagnetic radiation include fluorescent tags, such as fluorosceinisothiocyanate (FITC), Texas Red; Cyanin 5 and Cyanin 5.5; and otherfluorophores; electrochemiluminescent tags such as ruthenium trisbipyridyl salts, chemiluminescent tags such as CN, HF, HCF, and HCHO,and biochemiluminescent tags such as luciferase, luminol. Examples ofbioluminescent proteins include fusion proteins containing GFP (see,e.g., U.S. Pat. No. 5,958,713) or luciferase, aequorin and obelin (see,e.g., U.S. Pat. No. 5,683,888). See also, U.S. Pat. No. 5,656,207, andreferences cited therein, discussing the use of chemiluminescentmolecules, including acridinium and related compounds (e.g.phenanthridinium compounds), phthalhydrazides and related compounds(e.g. naphthalhydrazides), oxalate esters and related compounds and alsostabilized dioxetanes and dioxetanones. Additional examples of these arewell known in the art such as the disclosures of, and referencescontained in, U.S. Pat. Nos. 6,117,643 (bioluminescent species),6,008,057 (fluorescent species); 4,383,031 (chemiluminescent andenzyme-catalzyed fluorescent species); and 6,316,180(electrochemiluminescent species). Additional means of detection includecolorimetric endpoint detection. In a preferred embodiment, to enhancesignal-to-noise ratio of the electromagnetic radiation, CY3 fluorescencemay be used. It has been observed that beyond a certain packing density,CY3 (absorbs at 550 nm and exhibits emission maxima at 570 nm) emissionis amplified significantly (unlike CY5 which has been observed to havethe opposite, quenching effect). (J. B. Randolph et al., Nucleic AcidsResearch, 25 (1997) 2923-2929; H. J. Gruber et al., BioconjugateChemistry, 11 (2000) 696-704). The creation of an optically readablecode in a surface could also be achieved by inducing/modulating avariable strain in the material (in particular certain conductingpolymers) where polymer sections subjected to a certain strain providedifferent responses to a given optical excitation.

III. Surface Functionalization/Modification.

Functionalization or modification of the surface of the particle as usedherein refers to providing a means for covalently attaching samplemolecules to the surface of the particles. Surface functionalization ormodifications may include providing functional binding moieties on thesurface of the material including, but not limited to, chemical moietiessuch as carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal,hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate,acrylamide, substituted or unsubstituted thiol, halogen, substituted orunsubstituted amine, protected amine, hydrazide, protected hydrazide,succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone,allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, silyl,siloxyl, disiloxyl, mesyl, tosyl, and glyoxal moieties. Additionally,the functional binding moiety can comprise providing a “cationic moiety”on the surface, which comprises any positively charged species capableof electrostatically binding to negatively charged sample molecules,such as polynucleotides. Examples of cationic moieties include, but arenot limited to, polycations such as polylysine (e.g., poly-L-lysine),polyarginine, polyornithine, spermine, basic proteins such as histones,avidin, protamines, and modified albumins (e.g., N-acylurea albumin).The functional binding moiety may further include such reagents asantibodies, biotin, avidin, Ni-NTA to bind epitopes, botinylatedmolecules, hexahistidine tagged molecules, serum or collagen.

In the practice of the invention, the material from which the particlesare made can be either modified to contain the functional bindingmoieties or may contain such functional binding moieties as inherentproperties of the material. Methods of modifying the particle materialsdescribed above to contain such functional binding moieties are known tothose of ordinary skill in the art, with references dating back toMerrifield's description of solid-phase synthesis (R. B. Merrifield, J.Am. Chem. Soc. 85 (1963) 2149). Such functional binding moieties can becleavable but are preferably non-cleavable. Technologies developed forattaching compounds to solid substrates in combinatorial chemistrytechniques and solid phase peptide synthesis and linkers used to attacholigonucleotides to support materials used in chip-based systems areequally applicable here. Examples of such technologies are discussed inU.S. Pat. Nos. 6,362,009; 6,355,490; 6,352,828; 6,258,454; 6,147,159;6,248,540; 6,034,775; 6,291,669; 6,242,583; 6,232,066, and 6,057,456 andin the references disclosed therein.

In a preferred embodiment, surface functionalization or modification isconducted via an atmospheric plasma treatment using either a hydrazineor ammonia plasma, to attach amine functional binding moieties. Theprocess is preferably conducted on polymeric material. This process canbe optimized using modified gas flows and pressure in order to ensurehomogeneous coverage of the filament surface with NH₂. In this method,gas molecules are accelerated and diffuse towards the target surfaceunder the influence of electric and/or magnetic fields. Molecularbombardment knocks fragments of low molecular weight materials such aswater, adsorbed gases and polymer fragments off the surface of thematerial to expose a fresh, clean surface. At the same time, a certainpercentage of the reactive components of the plasma gas mixture havesufficient energy to bond to the freshly exposed surface resulting inthe changing of the chemistry of the surface and imparting the desiredfunctionality (e.g., primary amines attached via covalent bonds). Thisprocess typically produces an aminated surface layer less than 1 μmthick and tolerates solvents such as acetonitrile and strong acids.Indeed, all the conditions imposed by phosphoramidite oligonucleotidesynthesis can be tolerated by the coating. Hydrazine and ammonia plasmasare good candidates for surface functionalization since it may be usedwith a wide range of polymers including polyesters, polycarbonates, andpolyamides.

It is desirable that the functional binding moieties are added in a wayto form a homogeneous, dense, coverage on the surface of the material inorder to maximize binding of sample molecules to the resultant particleand, thereby, maximize an electromagnetic signal emanating from anattached electromagnetic emitting species. The layer should ideally notbe too dense so as to cause steric hindrance problems. For example, whenthe sample molecule is nucleic acid and the particles are used to detectand identify binding in a sequencing reaction, steric hindrance mayobstruct target nucleic acid during the binding process resulting indelayed or non-binding in a sample that otherwise could have bound.

IV. Sample Attachment.

Functionalization or modification provides a means of readily attachinga wide range of molecules to the surface of such materials which in turnprovides for a wide applicability of the particles made from thematerials. Such molecules are referred to herein as “probes” and includeall substances with an affinity to target molecules or compounds whosepresence, activity and/or amount in solution is desired to be determinedand which have an affinity for a given probe. The “target” molecules canbe man-made or naturally-occurring substances. Examples include, but arenot limited to, small molecules, dyes, carbohydrates, lipids, cellproducts, receptors, ligands, agonists or antagonists which bind tospecific receptors; polyclonal antibodies, monoclonal antibodies andantisera reactive with specific antigenic determinants such as onviruses, cells or other materials; drugs; nucleic acids orpolynucleotides, including mRNA, tRNA, rRNA, oligonucleotides, DNA,viral RNA or DNA, ESTs, cDNA, PCR-amplified products derived from RNA orDNA, and mutations, variants or modifications thereof; proteins such asenzymes, substrates for enzymes; peptides; cofactors; sugars;polysaccharides; cells; cellular membranes; organelles; viruses;liposomes; microparticles; micelles; chemokines; lymphokines, and othersubstances which can be complexed, covalently bonded, or crosslinkedwith these substances described. As used herein, the terms nucleic acid,polynucleotide, polynucleic acid and oligonucleotide are interchangeableand include those species having normal ribose-phosphate backbones orbackbones altered to enhance their properties as to attachment oflabels, stability and half-life of such molecules.

The term “probe” as used herein refers to any substance, such as amolecule, that can be specifically recognized by a particular target.The types of potential probe/target or target/probe binding partnersinclude receptor/ligand; ligand/antiligand; nucleic acid polynucleotide)interactions, including DNA/DNA, DNA/RNA, PNA (peptide nucleicacid)/nucleic acid; enzymes, other catalysts, or other substances, withsubstrates, small molecules or effector molecules; and the like.Examples of such probes include, but are not limited to, organic andinorganic materials or polymers, including metals, chelating agents orother compounds which interact specifically with metals, plastics,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones, hormone receptors, lipids, phospholipids,proteins, peptides, enzymes, enzyme substrates, cofactors, drugs,lectins, sugars, nucleic acids (such as defined above),oligosaccharides, polyclonal and monoclonal antibodies, single chainantibodies, or fragments thereof. Probe polymers can be linear orcyclic. Any of the substances described above as “probes” can also serveas “targets,” and vice-versa.

In a preferred embodiment, oligonucleotides are attached atfunctionalized zones of the material surface comprising linear primaryamines. The oligonucleotides can be generally attached to the particlesas follows. The amines carry a positive charge at neutral pH, allowingattachment of native DNA through the formation of ionic bonds with thenegatively charged phosphate backbone. Electrostatic attachment can besupplemented by treatment of the polymer surface with ultraviolet light,which induces free radical-based coupling between oligonucleotides andcarbon on the alkyl amine. The combination of electrostatic bonding andnon-specific covalent attachment links native DNA to the substratesurface in a stable manner. This method may be used for the attachmentof whole unmodified DNA, ideally those greater than 30-mers, or a slightvariant whereby base-by-base synthesis from an amine functionalizedsurface can be conducted in-situ, as described by Elder et al.,“Antisense Oligonucleotide Scanning Arrays”, published in DNAMicroarrays: A Practical Approach, Editor: M. Schena, (Oxford UniversityPress, 1999, pp. 77-99). Thus, the desired oligonucleotide sequences canbe built up as required to form n-mers on demand in a number of processsteps equal to n, without incurring additional retooling costs.Sequences are, therefore, fully user-determined and fully traceable.

V. Coding the Particles.

The coding mechanism used in the present invention relies on attachmentof sample molecules to select areas or portions of the surface of theparticles. Since the sample molecules readily attach to the surface ofthe particle having a functionalized binding moiety (i.e., having —NH₂groups thereon) the invention provides for spatially distributed codesby selectively modifying the surface of the particles to allow forbinding only in certain portions or zones of the material. Thisselective modification can be accomplished by selectively modifying orfunctionalizing the surface only in certain defined portions or zones,or alternatively by fully modifying or functionalizing the surface ofthe material and then removing the functional binding moieties incertain defined portions or zones. Thus, when a binding event occursbetween target and probe in the functionalized zones with concomitantemission of electromagnetic radiation, a detectable distinction betweenthe functionalized and differentially-functionalized zones occurs,revealing the spatially distributed code of the particle, andidentifying the binding of target and probe. As used herein, the term“differentially-functionalized zones” refers to zones of the particlesurface that have a different functionalization than the“functionalized” zones. For example, the differentially functionalizedzone can have different functional binding moieties than thefunctionalized zones such that the differentially functionalized zonesdo not preferentially bind probes and/or targets and/or electromagneticradiation emitting species. Alternatively, thedifferentially-functionalized zones can comprise no functionalization.

In the practice of the invention, the code can be formed by an orderedor random arrangement of the functionalized and differentiallyfunctionalized zones on the surface of the material. For example, thezones can be arranged as bars, bands, holes, bulls eyes, zebra stripes,spots, finger prints, and variations and combinations thereof.

In a preferred embodiment, coding is in the form of a series offunctionalized bands interspersed with differentially-functionalizedbands on the particle. This can be accomplished, for example, byexposing particular areas of the surface of the material with a laser,e.g., a UV-excimer laser, prior to the filament being cut intoparticles. The same result could be achieved by exploiting the fact thatpolymer extrusion processes can incorporate coatings that blockultraviolet light. A photo-modifiable polymer could be substituted forsuch a coating. The idea here would be to use the polymer as a mask,removing sections with a suitable wavelength light source prior toplasma treatment. In both embodiments, the laser/light source can beprogrammed to expose the areas of the material which allow a series offunctionalized bands of material and differentially-functionalizedsurface zones (together depicting the codes) for each particle. This iscontinually accomplished at the filament stage where the filament isadvanced past the laser source and rotated so as to ensure correct widthand depth of bands. Thereafter, a portion of the filament equal to thelength of a particle is cut by the laser yielding an optically readable,coded, cylindrical particle having a predetermined length.

In a preferred embodiment, the particle is formed in the shape of a rodof material and the coding of the particle is in the form of a series ofbands of functionalized binding moieties alternating with bandscontaining differentially-functionalized binding moieties. The codes areread by utilizing the electromagnetic radiation produced by theelectromagnetic radiation emitting species attached at thefunctionalized or modified areas. For example, when the electromagneticradiation is a fluorescent emission produced by a successful binding ofsample oligonucleotide to complementary oligonucleotide probes,fluorescent emission in the functionalized areas acts to illuminatethese areas so they can be read by, for example, fluorescence slitprofiling. In essence, because the binding event only occurs in theareas that are functionalized bands and not between them, theelectromagnetic radiation emission may envisioned as a binary aidesimilar to bar-coding with the functionalized zone or band representing1 and an differentially-functionalized zone or band representing 0. Inthis embodiment, if a particle has a length of, e.g., 130 μm and eachband is, e.g., 5 μm in width, then a total of 26 bands could beproduced, i.e. 26 bits. This would yield 2²⁶ or 67,108,864 possibledifferent codes. In such an embodiment, not all the bits are typicallyused for codes but are reserved for error checking. Thus the number ofpossible codes required would depend on the error checking schemechosen. Using Bi-Phase or Manchester Coding, for example, and ensuringan even power distribution across the coded area, only 50% of the bitswould be used for coding. Thus, 2¹³ or 8192 different codes would beavailable for the particles. Such a code-space is particularlyapplicable to a wide range of sequencing applications including SNPapplications which typically require somewhere between 100 and 5000different codes.

The width of the bands are generally between about 1 μm and 50 μm, andmost preferably between approximately 1 μm and 5 μm.

VI. Particle Based Assay Methodology.

The particles of the invention can be applied to various liquid-basedassays to perform quantitative, semi-quantitative, qualitative, orratiometric determinations of targets in solution. Such assays include,but are not limited to SNP detection, hybridization assays, enzymaticextensions and immunoassay techniques such as sandwich assays,competitive assays, and displacement assays. Thus, for example, theinvention provides a method for quantitatively assaying the binding ofthe probe with its identification code and its corresponding target. Insuch a configuration, oligomers or binder proteins (for example, MABs)that serve as ‘probes’ would be attached to functionalized areas of theparticle surface. On exposure of the particle to sample containingtarget nucleotides or proteins (e.g., at time=0), mixing would ensuremass-transfer independent binding of target to probe coated particle.Assuming the nucleotides or binder protein to have a high affinity forthe target, and low cross-reactivity with others, the amount of targetmaterial bound at defined points in time would be a function of theconcentration of targets in the sample. For nucleotides, an attachedreporter fluorophore would provide information of its presence. Forproteins subsequent exposure to a suitable secondary antibody to which areporter fluorophore is attached would provide information of itspresence. The reporter would also reveal its concentration relative toan established standard (quantitative or semi-quantitative), or,relative to other target proteins present in the same sample but boundto different particles (ratiometric).

In one embodiment, the invention provides a method of determining thesequence of an unknown oligonucleotide species in a solution comprisingproviding a labeled oligonucleotide library of particles according tothe invention, wherein the oligonucleotide sequence of eacholigonucleotide attached to each particle in the library and thecharacteristic electromagnetic emission intensity profile of eachparticle in the library is stored and correlated in a database, mixingthe solution with the labeled oligonucleotide library under conditionssufficient to permit the unknown oligonucleotide speices to hybridizewith corresponding particles of the labeled oligonucleotide library,exciting the particles to produce the characteristic electromagneticemission intensity profile corresponding to the particle(s) in thelibrary, detecting the characteristic electromagnetic emission intensityprofile and correlating it to the characteristic emission intensityprofile in the database, thereby determining the sequence of the unknownoligonucleotide species.

In the practice of this method the characteristic emission profile canbe detected by a charged coupling device (CCD) array, photodiode array,or photomultiplier tube. Typically a laser is used to excite theparticles, however other similar electromagnetic emission excitationdevice are equally applicable.

In the practice of the invention the unknown oligonucleotide species canbe, e.g., a single nucleotide polymorphism (SNP), cDNA cloned from RNAexpressed by a normal cell or cDNA cloned from RNA expressed by a cellthat has been subjected to a drug, toxic agent, or other chemicalsubstance.

In other embodiments, the invention also provides a method for detectinga genetic mutation in a PCR product amplified from a nucleic acid samplecontaining a target gene of interest, comprising the steps:

-   -   (a) selecting an oligonucleotide probe, said oligonucleotide        probe including a polymorphic site, said polymorphic site        including said genetic mutation or the wild type sequence found        at the analogous position of said genetic mutation in a wild        type target gene;    -   (b) coupling said oligonucleotide probe to one of a plurality of        particles of a library of particles according to to the        invention to form a labeled probe library;    -   (c) providing PCR products comprising the region of said target        gene amplified by PCR;    -   (d) mixing the labeled probe library and the PCR products;    -   (e) incubating said mixture under hybridization conditions,        wherein if said PCR products include said polymorphic site, said        PCR products bind to a particle(s) of said oligonucleotide probe        library;    -   (f) exciting the particles to produce the characteristic        electromagnetic emission intensity profile corresponding to the        particle(s) in the library,    -   (g) detecting the characteristic electromagnetic emission        intensity profile of the particle(s); and    -   (h) detecting said genetic mutation, or absence thereof, as a        function of the measured characteristic electromagnetic emission        intensity profile of the particle(s).

In another embodiment, the invention provides a method for determiningthe amount of a target compound in a test sample comprising the steps ofincubating a mixture of a test sample suspected of containing the targetcompound with at least one particle according to to the invention,wherein the particle(s) comprise a plurality of an antibody which isspecific for the target compound, under conditions appropriate to formtarget/antibody complexes, wherein the electromagnetic radiationemission occurs upon formation of the target/antibody complex, andmeasuring the amount of electromagnetic radiation present in saidmixture thereby determining the amount of target compound in said testsample.

The invention also provides an assay for the detection of bindingbetween a probe and a target comprising contacting the probe with asolution suspected of containing the target, wherein the probe isattached to a particle which emits electromagnetic radiation uponbinding between probe and target and emits electromagnetic radiation asa means of identifying the particle in solution.

In one embodiment of the assay, the particle comprises a singleelectromagnetic radiation emitting species that emits both theelectromagnetic radiation upon binding between probe and target and theelectromagnetic radiation as a means of identifying the particle insolution. In another embodiment of the assay, the particle comprisesdifferent electromagnetic radiation emitting species to emit theelectromagnetic radiation upon binding between probe and target and toemit the electromagnetic radiation as a means of identifying theparticle in solution. In the practice of the assay, the electromagneticradiation emitted as a means of identifying the particle is emitted as apattern of electromagnetic radiation, wherein the pattern is formed byattaching the electromagnetic radiation emitting species tofunctionalized zones on the surface of the particle spatiallydistributed among differentially functionalized zones on the surface ofthe particle that do not contain the electromagnetic radiation emittingspecies.

Preferred embodiments of particle based assay methods are depicted inFIG. 2, in which a fluorescent-labeled sample protein as shown is mixedwith the coded particles and FIG. 3 in which a fluorescent-labeledsample DNA is mixed with the coded particles. After a predeterminedperiod of time, the particles are flowed past a laser source of the flowcytometer as shown in a sandwich assay on FIG. 2 (or DNA withfluorophore in FIG. 3) The laser excites the fluorophore which hasattached itself to probe/target, either monoclonal antibody in FIG. 2 orcDNA/N-mer in FIG. 3 on the particle, whereby any binding is indicatedby a high intensity fluorescent emission detected by a CCD camera. Thecode may then be read by fluorescent slit profiling, in which thefluorescent bands are viewed as a series of peaks (corresponding to thebands having an attached fluorophore) and troughs (areas between bands)whose width and separation is characteristic of the particle's code. TheCCD outputs the resultant fluorescent energy waveform to the computercontaining the sample-particle database (library). The computer thancompares the distances between the peaks of the waveform, to thedistances between bands of cataloged particles to find the particularcataloged particle. Once the particle is found in the database, theunknown protein or DNA sample is then determined.

EXPERIMENTAL DETAILS Example 1 Particle Material SurfaceFunctionalization

A polypropylene filament was subjected to the radio frequency (RF)discharge method which involves exposing the polymer to an ammoniaplasma, using substantially the method as disclosed by BeckmanInstruments (See, Matson, R. S., Rampal J. B. and Coassin P. J.,Biopolymer Synthesis on Polypropylene Supports, Anal. Biochem., (1994)217, 306-310). The process is automated, robust, the whole surface isexposed, and many filaments can be treated at the same time. The plasmaprocess parameters were as follows:

gas: ammonia,

chamber pressure: 25-30 Pa

gas flow rate: 35 cubic centimeters per second

treatment time: 90 seconds.

X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy(SEM) surface characterization techniques were used to analyze theresults of the treatment process. The aim of the XPS was to characterizethe surfaces of two filament samples, by determination of the surfaceconcentration and chemical state of detectable elements. Both samplescontained carbon (as C—(C,H), C—(O,N), and C═O), oxygen, nitrogen (asC—N and R₄—N⁺), and phosphorus (as PO_(x)). The aminated filament alsocontained carbon (as (O,N)—C═O), chlorine (as Cl⁻), and silicon (assilicon and/or silicate), while having an increased concentration ofoxygen and a three-fold increase in nitrogen relative to the controlsample. Quantification of the elements was accomplished by using theatomic sensitivity factors for a Physical Electronics Model 5700LSciESCA Spectrometer analytical conditions: X-ray source (monochromaticAl); source power (350 W); analysis region (2 mm×0.8 mm); exitangle)(50°).

The plasma process creates precursor —NH₂ and —NH groups which diffusethrough the chamber to the polymer substrate. Successful surfaceamination was confirmed by the attachment of primary amine specific FITC(FIGS. 4(A) and 4(B)). Both plasma treated and non plasma treatedsurfaces were exposed to FITC. FITC was not observed to bind on the nonplasma treated surface. Images were taken with a fluorescencemicroscope. FIG. 4(A) shows the polypropylene surface prior to plasmatreatment; FIG. 4(B) shows the polypropylene surface following plasmatreatment (each is further depicted in a drawing below the correspondingphotomicrograph). The images were captured by an Olympus IMT-2 invertedmicroscope coupled so a 75-W Xenon arc lamp and fitted with aninterference filter and an IR filter. The image was formed on aPrinceton Instruments thermoelectrically-cooled CCD camera (ModelTE/CCD-500B with an ST-130 controller). The 518×384 pixel images had aresolution of 1.2 μm and a dynamic resolution of 16-bits. Colors wereachieved by a combination of suitable filtering and mathematicalmodeling. Excitation wavelengths in the 480-500 nm range were used.

FIG. 4(A) demonstrates both the low-background of polypropylene (i.e.,low auto-fluorescence) as well as the fact that amine-specific FITC doesnot bind to it. Low auto-fluorescence is important in order to reducebackground noise during fluorescence profiling (when the code is being‘read’). Polymers (such as polyamide) tend to have a highauto-fluorescence, so its low level is a significant advantage here.

Example 2 Laser Ablation of Particles

Polypropylene filament was etched by a 248 nm UV-excimer laser to ablateareas of functionalized polymer in order to expose underlying inert and,therefore, differentially-functionalized, material. A Potomac LMT-4000was used to create grooves of 10 μm wide The system contained a PotomacTGX-1000 KrF (248 nm) excimer laser configured for focused operation atthe polymer surface. The laser beam was apertured in order to achieve a5 μm spot size at the surface. The maximum pulse repetition rate was 2KHz with a maximum pulse energy of 45 μJ. The photon energy of about 4to 7 eV at UV wavelengths is about 30 to 40 times greater than that ofCO₂ laser radiation. Consequently, there is a vast difference inlaser-material interactions. Most organic polymers absorb strongly inthe UV, and laser ablation in this spectral range produces sharp edgesand a lack of charring when contrasted with that performed bylong-wavelength devices such as CO₂ lasers. Since the absorptioncoefficient for UV light is very high for most materials, the energy isabsorbed in a very thin surface layer. Subsequent attachment offluorescent substances as described in Example 1 to the non-ablatedactive areas will reveal the code.

Having presented the invention in view of the above describedembodiments, various alterations, modifications, and improvements areintended to be within the scope and spirit of the invention. Theforegoing description is by way of example only and is not intended aslimiting. The invention's limit is defined only in the following claimsand the equivalents thereto.

1. A particle comprising a surface, wherein a portion of the surface orsub-surface is capable of emitting a first electromagnetic radiation andanother portion of the surface is capable of emitting a differentialelectromagnetic radiation, and wherein the arrangement of said portionsof the surface defines a spatially distributed code for identifying theparticle. 2.-7. (canceled)
 8. The particle of claim 1, wherein theelectromagnetic radiation is derived from non-optical excitation of theparticle.
 9. The particle of claim 8, wherein the non-optical excitationis chosen from the group consisting of electrical, magnetic, physical,chemical, biological, electrochemical, and combinations thereof. 10.-15.(canceled)
 16. The particle of claim 1, wherein the particle is formedfrom a material chosen from the group consisting of polymers,composites, inorganics, natural products, and combinations thereof. 17.(canceled)
 18. The particle of claim 16, wherein the composites arechosen from the group consisting of glass fiber composites, carbon fibercomposites, and combinations thereof.
 19. The particle of claim 16,wherein the natural products are chosen from the group consisting ofsilk, wax, rubber, resins, and combinations thereof.
 20. (canceled) 21.A particle comprising a surface wherein the surface comprises at leastone modified portion comprising a plurality of functional bindingmoieties and at least one unmodified portion which is substantially freeof functional binding moieties, wherein the modified portion(s) furthercomprise an electromagnetic radiation emitting species, and wherein thearrangement of modified and unmodified portions on the surface forms apattern amenable to detection.
 22. The particle of claim 21, wherein thepattern defines a spatially distributed code for identifying theparticle.
 23. The particle of claim 21, wherein the electromagneticradiation emitting species is attached to at least one of the pluralityof said functional binding moieties.
 24. The particle of claim 21,wherein the electromagnetic radiation emitting species is attached to acompound attached to at least one of the plurality of said functionalbinding moieties.
 25. The particle of claim 21, wherein one or morefunctional binding moieties comprise a probe to which is bound a targetspecies and wherein the electromagnetic radiation emitting species isattached to the target species.
 26. The particle of claim 21, whereinthe electromagnetic radiation emitting species is chosen from the groupconsisting of fluorescent, electrochemiluminescent, chemiluminescent,and biochemiluminescent species.
 27. The particle of claim 26, whereinthe fluorescent species is chosen from the group consisting offluoroscein isothiocyanate (FITC), Texas Red, Cyanin 5 and Cyanin 5.5.28. The particle of claim 26, wherein the electrochemiluminescentspecies is a ruthenium tris bipyridyl salt.
 29. The particle of claim26, wherein the chemiluminescent species is chosen from the groupconsisting of CN, HF, HCF, HCHO, acridinium compounds, phthalhydrazides,oxalate esters, stabilized dioxetanes and dioxetanones.
 30. The particleof claim 26, wherein the biochemical luminescent species is chosen fromthe group consisting of green fluorescent protein (GFP), luciferase,luminol, aequorin and obelin.
 31. A method of manufacturing a particlehaving an identifying code comprising providing a particle with afunctionalized surface which comprises functional binding moieties andselectively removing a plurality of the functional binding moieties fromthe surface to create a pattern of functionalized anddifferentially-functionalized zones on the surface.
 32. The method ofclaim 31, further comprising the step of contacting the particle with aplurality of electromagnetic radiation emitting moieties underconditions appropriate to allow for the covalent binding of theelectromagnetic radiation emitting moieties to the plurality of thefunctionalized binding moieties.
 33. The method of claim 32, wherein theparticle is contacted with the electromagnetic radiation emittingmoieties after the selective removal of a plurality of functionalbinding moieties.
 34. The method of claim 32, wherein the particle iscontacted with the electromagnetic radiation emitting moieties beforethe selective removal of a plurality of functional binding moieties. 35.The method according to claim 31, wherein the particle includes across-section comprising a geometric shape.
 36. The method according toclaim 31, wherein the particle includes a substantially cylindricalcross-section, and wherein the functionalized zones and thenonfunctionalized zones are positioned substantially around thecircumference of the cross-section.
 37. The method according to claim31, wherein the selective removal of functional binding moieties fromthe surface comprises contacting the particle with laser light.
 38. Themethod according to claim 37, wherein the laser light is produced by aUV-excimer laser.
 39. The method according to claim 37, wherein thelaser light is produced by a pulsed laser.
 40. A method of manufacturinga particle having an identifying code comprising providing a particlehaving a differentially-functionalized surface and selectively adding aplurality of functional binding moieties to the surface to create apattern of functionalized and differentially-functionalized zones on thesurface.
 41. The method of claim 40, further comprising the step ofcontacting the particle with a plurality of electromagnetic radiationemitting moieties under conditions appropriate to allow for the covalentbinding of the electromagnetic radiation emitting moieties to theplurality of the functionalized binding moieties.
 42. The methodaccording to claim 40, wherein the particle includes a cross-sectioncomprising a geometric shape.
 43. The method according to claim 40,wherein the particle includes a substantially cylindrical cross-section,and wherein the functionalized zones and the nonfunctionalized zones arepositioned substantially around the circumference of the cross-section.44. An article of manufacture having a substantially cylindrical shapewith a diameter between about 5 μm and 200 μm, and a length betweenabout 10 μm and 2000 μm, wherein the surface of the particle comprisesat least one substantially circumferential functionalized zone, at leastone substantially circumferential differentially-functionalized zonebetween the end of the particle and the functionalized portion orbetween functionalized portions, and a combination of the width(s) ofthe functionalized portion(s) and the width(s) of thedifferentially-functionalized zone(s) establishes a code for identifyingthe particle.
 45. A labeled oligonucleotide library comprising aplurality of oligonucleotide compounds attached to a plurality ofparticles according to claim
 1. 46. The labeled oligonucleotide libraryof claim 45, wherein each particle contains a single species ofoligonucleotide compound.
 47. The labeled oligonucleotide library ofclaim 45, wherein the oligonucleotide compounds are syntheticoligonucleotides.
 48. A particle according to claim 21 furthercomprising a plurality of oligonucleotide compounds attached to theplurality of functionalized binding moieties.
 49. The particle of claim48 wherein the oligonucleotide compounds are of a single known speciesof oligonucleotide compound.
 50. A method of determining the sequence ofan unknown oligonucleotide species in a solution comprising providing alabeled oligonucleotide library of claim 45, wherein the oligonucleotidesequence of each oligonucleotide attached to each particle in thelibrary and the characteristic electromagnetic emission intensityprofile of each particle in the library is stored and correlated in adatabase, mixing the solution with the labeled oligonucleotide libraryunder conditions sufficient to permit the unknown oligonucleotide spciesto hybridize with corresponding particles of the lableledoligonucleotide library, exciting the particles to produce thecharacteristic electromagnetic emission intensity profile correspondingto the particle(s) in the library, detecting the characteristicelectromagnetic emission intensity profile and correlating it to thecharacteristic emission intensity profile in the database, therebydetermining the sequence of the unknown oligonucleotide species.
 51. Themethod according to claim 50, wherein the characteristic emissionprofile is detected by a charged coupling device (CCD) array, photodiodearray, or photomultiplier tube.
 52. The method according to claim 50,wherein a laser is used to excite the particles.
 53. The methodaccording to claim 50, wherein a flow cytometer is used to mix theparticle library with the solution containing the unknownoligonucleotide species.
 54. The method according to claim 50, whereinthe unknown oligonucleotide species is a single nucleotide polymorphism(SNP).
 55. The method according to claim 50, wherein the unknownoligonucleotide species is cDNA cloned from RNA expressed by a normalcell.
 56. The method according to claim 50, wherein the unknownoligonucleotide species is cDNA cloned from RNA expressed by a cell thathas been subjected to a drug, toxic agent, or other chemical substance.57. A method for detecting a genetic mutation in a PCR product amplifiedfrom a nucleic acid sample containing a target gene of interest,comprising the steps: (a) selecting an oligonucleotide probe, saidoligonucleotide probe including a polymorphic site, said polymorphicsite including said genetic mutation or the wild type sequence found atthe analogous position of said genetic mutation in a wild type targetgene; (b) coupling said oligonucleotide probe to one of a plurality ofparticles of a library of particles according to claim 45 to form alabeled probe library; (c) providing PCR products comprising the regionof said target gene amplified by PCR; (d) mixing the labeled probelibrary and the PCR products; (e) incubating said mixture underhybridization conditions, wherein if said PCR products include saidpolymorphic site, said PCR products bind to a particle(s) of saidoligonucleotide probe library; (f) exciting the particles to produce thecharacteristic electromagnetic emission intensity profile correspondingto the particle(s) in the library, (g) detecting the characteristicelectromagnetic emission intensity profile of the particle(s); and (h)detecting said genetic mutation, or absence thereof, as a function ofthe measured characteristic electromagnetic emission intensity profileof the particle(s).
 58. A method for determining the amount of a targetcompound in a test sample comprising the steps of incubating a mixtureof a test sample suspected of containing the target compound with atleast one particle according to claim 21, wherein the particle(s)comprise a plurality of an antibody which is specific for the targetcompound, under conditions appropriate to form target/antibodycomplexes, wherein the electromagnetic radiation emission occurs uponformation of the target/antibody complex, and measuring the amount ofelectromagnetic radiation present in said mixture thereby determiningthe amount of target compound in said test sample.
 59. An assay for thedetection of binding between a probe and a target comprising contactingthe probe with a solution suspected of containing the target, whereinthe probe is attached to a particle which emits electromagneticradiation upon binding between probe and target and emitselectromagnetic radiation as a means of identifying the particle insolution.
 60. The assay of claim 59, wherein the particle comprises asingle electromagnetic radiation emitting species that emits both saidelectromagnetic radiation upon binding between probe and target and saidelectromagnetic radiation as a means of identifying the particle insolution.
 61. The assay of claim 59, wherein the particle comprisesdifferent electromagnetic radiation emitting species to emit saidelectromagnetic radiation upon binding between probe and target and toemit said electromagnetic radiation as a means of identifying theparticle in solution.
 62. The assay of claim 59, wherein theelectromagnetic radiation emitted as a means of identifying the particleis emitted as a pattern of electromagnetic radiation, wherein thepattern is formed by attaching the electromagnetic radiation emittingspecies to functionalized zones on the surface of the particle spatiallydistributed among differentially functionalized zones on the surface ofthe particle that do not contain the electromagnetic radiation emittingspecies.
 63. The assay of claim 59, wherein the first electromagneticradiation is of a wavelength from ultraviolet through to infra-red. 64.The assay of claim 59, wherein the first electromagnetic radiation is ofa wavelength between 400 nm to 1 μm.
 65. A kit for the detection ofbinding between a probe and a target comprising at least one particle asdefined in claim 1 and a flow cytometric device.